Longevity factor klotho enhances cognition in aged nonhuman primates

Abstract

Cognitive dysfunction in aging is a major biomedical challenge. Whether treatment with klotho, a longevity factor, could enhance cognition in human-relevant models such as in nonhuman primates is unknown and represents a major knowledge gap in the path to therapeutics. We validated the rhesus form of the klotho protein in mice showing it increased synaptic plasticity and cognition. We then found that a single administration of low-dose, but not high-dose, klotho enhanced memory in aged nonhuman primates. Systemic low-dose klotho treatment may prove therapeutic in aging humans.

Fig. 1. Rhesus klotho is biologically active and increases serum KL levels in aged rhesus macaques.

a, Paradigm of hippocampal LTP recordings from young male mice treated with either vehicle (Veh) or rhesus KL (10 μg kg⁻¹, s.c.). b, Field excitatory post-synaptic potential (fEPSP) recordings from acute hippocampal slices of mice (age 3 months) (n = 8 biologically independent slices from three mice for Veh; ten biologically independent slices from three mice for rhesus KL). c, Average fEPSP slope of the last 10 min of recordings from slices of mice (same as b). ***P < 0.0001 (two-tailed Student’s t-test); if biological unit is mouse, **P = 0.0039 (two-tailed Student’s t-test). d, Paradigm for testing male mice (age 4 months) in the small Y maze following treatment with Veh or rhesus KL (10 μg kg⁻¹, s.c.). e, Percent alternations of mice (n = 28 mice, Veh; n = 29 mice, KL). Two independent cohorts combined. *P = 0.0243 (two-tailed Student’s t-test; *P = 0.0249 KL effect, accounting for cohort by linear regression two-tailed t-test). f, Paradigm for measuring serum KL levels in young male mice (age 4 months) 4 h following treatment with Veh or mouse KL (10 μg kg⁻¹, s.c.). g, Relative KL serum levels from nontransgenic (NTG) and KL transgenic overexpressing (KL TG) mice alongside serum from young mice (age 4 months) 4 h following treatment with Veh or mouse KL (10 μg kg⁻¹, s.c.) (n = 5 mice in each group of NTG, Veh, KL; n = 7 mice, KL TG). **P < 0.0001 versus NTG (two-tailed t-test), ***P = 0.0036 versus Veh (two-tailed t-test). h, Paradigm for measuring serum KL levels in aged rhesus macaques 4 h following treatment with Veh or varying doses of rhesus KL. i, Relative KL serum levels of aged rhesus macaques at baseline and following varying doses of KL (baseline, n = 19 monkeys; n = 14 female, n = 5 male; KL dosing, n = 3 monkeys in each group at 0.4 and 2 μg kg⁻¹; n = 4 monkeys in each group at 20 and 30 μg kg⁻¹; n = 5 monkeys at 10 μg kg⁻¹; all female except one male at 10 μg kg⁻¹). Values relative to baseline. *P = 0.0479 (KL 20 μg kg⁻¹) and *P = 0.0508 (KL 30 μg kg⁻¹) (one-tailed, one-sample t-tests versus KL 10 μg kg⁻¹); **P = 0.0046 and ***P < 0.0001 versus baseline (two-tailed, Bonferroni-corrected). Data are represented as mean ± s.e.m. Source data

Fig. 2. Rhesus KL enhances cognition in aged rhesus macaques.

a, Paradigm for testing aged rhesus macaques (age 15–28 years; n = 18 monkeys; 13 females and 5 males) in a spatial delayed memory task following treatment with Veh or rhesus KL (Supplementary Table 1 shows testing in each monkey). b, Diagram of the SDR cognitive task. Monkey is shown food reward (cue phase), screen is lowered (delay) and then screen is raised with all wells covered (test phase). Remembering location of hidden reward in NML and HML (more wells and longer delays) was measured. c, Percent correct choices by monkeys, representing spatial and working memory, in HML task at baseline (n = 19 sessions from 18 monkeys; 13 females and 5 males), at 4 h following treatment with Veh (n = 26 sessions from 18 monkeys; 13 females and 5 males) or rhesus KL (10 μg kg⁻¹) (n = 11 sessions from 9 monkeys; 6 females and 3 males). **P = 0.0077 versus Veh (linear mixed-model ANOVA and Satterthwaite’s t-tests using sessions, two-tailed). Bars represent mean ± s.e.m. of test sessions; points are mean performance of each monkey. d, Percent correct choices by monkeys in the HML task 4 h and 14–23 d after a single injection with Veh or rhesus KL (10 μg kg⁻¹). n, same as Fig. 1c. **P = 0.0077 (4 h), **P = 0.0035 (14–23 d) versus Veh (linear mixed-model ANOVA and Satterthwaite’s t-tests using sessions, two-tailed). Points indicate sessions. Filled circles are mean ± s.e.m. of sessions. Dashed line shows mean Veh performance. e, Percent correct choices by monkeys in NML task 1–14 d after baseline or vehicle treatment (n = 71 sessions from 17 monkeys; 12 females and 5 males) or rhesus KL (10 μg kg⁻¹) (n = 46 sessions from 9 monkeys; 6 females and 3 males), ***P = 0.0006 versus Veh + baseline (linear mixed-model ANOVA and Satterthwaite’s t-tests using session, two-tailed). Points show mean performance of each monkey. Bars represent mean ± s.e.m. of test sessions. f, Percent correct choices by monkeys in NML task averaged over the first and second weeks after baseline or Veh treatment (Ctl) (n, same as Fig. 1e) or rhesus KL (10 μg kg⁻¹ (n, same as Fig. 1e; 32 sessions between 1–7 d and 14 sessions between 8–14 d), **P = 0.0080 (1–7 d) and **P = 0.0021 (8–14 d) versus Veh + baseline (linear mixed-model ANOVA and Satterthwaite’s t-tests using sessions, two-tailed). Points indicate sessions. Filled circles indicate mean ± s.e.m. of sessions. Dashed line shows mean control performance. g, Percent increase in cognition in HML task at varying doses of rhesus KL treatment at 10 μg⁻¹ averaged over the course of testing (n = 11 sessions from 9 monkeys; 6 female and 3 male), 20 μg kg⁻¹ (n = 7 sessions from 7 monkeys; 5 female and 2 male) or 30 μg kg⁻¹ (n = 13 sessions from 13 monkeys; 9 females and 4 males) compared to Veh treatment (n = 26 sessions from 18 monkeys; 13 females and 5 males). **P = 0.0077 versus Veh (linear mixed-model ANOVA and Satterthwaite’s t-tests using sessions, two-tailed). h, Percent increase in cognition in NML task at varying doses of rhesus KL treatment at 10 μg kg⁻¹ averaged over the course of testing (n = 46 sessions from 9 monkeys; 6 female and 3 male), 20 μg kg⁻¹ (n = 31 sessions from 7 monkeys; 5 female and 2 male) or 30 μg kg⁻¹ (n = 40 sessions from 13 monkeys; 9 females and 4 males) compared to Veh + baseline (n = 71 sessions from 17 monkeys; 12 females and 5 males) ***P = 0.0006 versus Veh (linear mixed-model ANOVA and Satterthwaite’s t-tests using sessions, two-tailed). Source data

Extended Data Fig. 1. Rhesus klotho treatment of mice did not alter total arm entries in the Y maze, despite increasing % alternations.

Mice (n = 28 mice, Veh; n = 29 mice, KL; age=4 months, male) treated with Veh or Rhesus KL (s.c., 10 μg/kg) did not differ in their number of entries despite klotho-mediated increased % alternations (Related to Fig. 1e). Data are mean ± SEM. Source data

Extended Data Fig. 2. Time course of serum klotho levels following treatment of two aged rhesus macaques.

Following Rhesus KL injection (s.c., 30 μg/kg), blood was collected at 4 h, 28 h and 52hrs (n = 8 samples from 2 monkeys, female, ages 22 and 24 years). Assuming peak concentration at 4 h, putative klotho half-life was 29.5 h with predicted elimination in 6.15 days. Source data